EP0061857B1 - Test d'agglutination de particules - Google Patents

Test d'agglutination de particules Download PDF

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Publication number
EP0061857B1
EP0061857B1 EP82301265A EP82301265A EP0061857B1 EP 0061857 B1 EP0061857 B1 EP 0061857B1 EP 82301265 A EP82301265 A EP 82301265A EP 82301265 A EP82301265 A EP 82301265A EP 0061857 B1 EP0061857 B1 EP 0061857B1
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EP
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Prior art keywords
hapten
particles
antigen
assay
agglutination
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Expired
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EP82301265A
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German (de)
English (en)
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EP0061857A1 (fr
Inventor
Pierre Lucien Masson
Daniel Collet-Cassart
Carl Gustav Magnusson
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International Institute of Cellular and Molecular Pathology
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International Institute of Cellular and Molecular Pathology
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/805—Optical property
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/815—Test for named compound or class of compounds
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/821—Chemistry: analytical and immunological testing involving complement factors or complement systems
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/825—Pretreatment for removal of interfering factors from sample

Definitions

  • This invention relates to a particle agglutination assay which is especially suitable for the analysis of haptens but which may also be used to assay antigens.
  • the term "antigen" excludes haptens, i.e. excludes small non-protein substances which are monovalent and have a molecular weight below about 1500.
  • a particle agglutination assay for antigens and haptens is described in our U.S. patent No. 4184849.
  • This assay uses two different particulate reagents which mutually agglutinate when mixed together, but whose agglutination is inhibited by the presence of the antigen or hapten under assay. Whilst this assay is very satisfactory in practice, it has the disadvantage of requiring two different particulate reagents, each specific to the particular assay being performed, and each being used in controlled amounts. The use of two such reagents increases the complexity of the overall procedure and is, from this point of view, undesirable.
  • Another type of particle agglutination assay which uses only one particulate reagent, is known for the assay of immune complexes (see Methods in Immunology, supra 133-134).
  • the sample containing immune complexes to be assayed
  • immunoglobulin G-coated latex particles and a limited amount of an agglutinator.
  • Competition for reaction with the agglutinator occurs between the latex-lgG and the free complexes, and the amount of complexes in the sample can be determined from the degree of agglutination of the particles.
  • This method of assay is very sensitive, rapid and precise.
  • a similar procedure could be used to assay antigens, by mixing the sample (containing the antigen under assay) with latex particles bearing the same antigen, and an excess of antibody. Both the free and the latex-bound antigen would then bind to antibody to form complexes, and by adding a limited amount of agglutinator, agglutination of the particles will occur to an extent dependent on the amount of antigen present in the original sample.
  • a particle agglutination assay for haptens which involves the use of only one latex particle reagent, and which does not rely on the use of critical quantities of agglutinator.
  • affinity can surprisingly be markedly increased if the hapten is first bound to a latex particle, e.g. conjugated to a carrier such as a protein or polymer, and then coupled to the latex, or coupled directly to the latex.
  • the strength of the bond then formed between the latex-bound complex and the agglutinator is, we have found, sufficient to cause two latex particles (each bearing complex) to become bound together by the agglutinator. This is in marked contrast to the effect of agglutinator on free hapten-antibody complexes, where the affinity between the agglutinator and the complexes is too weak to cause any significant binding.
  • a hapten as follows.
  • the sample containing the hapten under assay is mixed with latex (or other particles) bearing the same hapten as is under assay or a specific analogue thereof, and a limited amount of antibody.
  • the free hapten and the latex-bound hapten compete for the limited quantity of antibody, forming complexes therewith, one portion of the antibody becoming bound to free hapten and another portion becoming bound to the latex-bound hapten.
  • An agglutinator is also included in the mixture and this causes agglutination only of those latex particles bearing the hapten-antibody complex.
  • the agglutinator does not bind to any significant extent with either the latex-hapten, or the free hapten, or the free hapten-antibody complexes.
  • the presence and extent of agglutination indicates the presence and amount of the hapten under assay.
  • the amount of agglutinator required is merely that amount necessary to cause sufficient agglutination of the latex-complex particles. Since, in fact, no other component of the hapten assay reaction mixture has any very significant binding capacity for the agglutinator, very little agglutinator is in practice necessary. Accordingly, the method of the invention has the advantage of very economic use of an expensive reagent in the assay of haptens. Further, the use of a critically determined amount of agglutinator is completely avoided, thus improving the reproducibility and accuracy of the assay results.
  • the competition reaction occurs not in respect of the agglutinator (which is inevitably of variable quality), but rather in respect of the antibody.
  • the production of reproducible antisera is a well established procedure, and hence the prior art problem is overcome.
  • the method of the invention is of particular utility in the assay of haptens, it may equally be used for the assay of antigens.
  • a method of assaying an antigen or a hapten in a liquid sample which comprises mixing with the liquid sample:
  • Such multivalent regions do not occur, of course, in dilute solutions of free hapten-antibody complex.
  • the hapten-antibody complexes are "concentrated", so providing an increased affinity for agglutinator.
  • a "limited amount" of antibo'dy is that quantity of antibody which when added to a latex-antigen or latex-hapten agglutination reaction mixture, causes less than 100% agglutination, a preferred value being from 40 to 80%, most preferably between 70 and 80% agglutination.
  • the concentration of latex-antigen or latex-hapten is itself preferably adjusted so that approximately 50% of the particles are agglutinated when added to a solution of approximately 50% of the expected assay range concentration of antigen or hapten, with the agglutinator in excess (i.e. ⁇ 10% change of agglutinator concentration causes no appreciable change in measured concentration of unagglutinated particles).
  • the method of the invention may be used for the assay of antigens or haptens and as such is of broad utility.
  • Particular haptens which can advantageously be assayed by the method include drugs and drug metabolites such as theophylline, barbitone, phenylhydantoin, digoxin, digitoxin and gentamicin; intracellular messenger such as cyclic AMP and cyclic GMP, prostaglandins and prostaglandin metabolites; hormones such as hypothalmic and pineal hormones, e.g. thyrotropin releasing hormone, gonadotropin releasing hormone, somatostatin, melatonin, substance P, neurotensin; pituitary hormones, e.g.
  • thyroid hormones e.g. thyroxine and triiodothyronine
  • hormones of the gastrointestinal tract e.g. gastrin, secretin, cholecystokinin and serotonin
  • pancreatic hormones e.g. glucagon and C-peptide
  • steroid hormones e.g. estradiol, progesterone, testosterone, aldosterone and cortisol
  • vitamins such as B12, folic acid, vitamin D metabolites
  • haptens of bacteriological or virological origin e.g. the glycolipid of leucosis virus.
  • the liquid to be assayed for a particular hapten will usually be of biological origin, e.g. human or animal body fluids such as blood serum, saliva or urine.
  • the particulate reagent will normally be latex particles as described hereinbefore, but other particles (whilst not preferred), such as agarose gel; Sephadex@ gel or bentonite, could be used.
  • the particles bear the same antigen or hapten as is under assay or a specific analogue thereof.
  • the preparation of antigen- or hapten-bearing particles is well known in the art.
  • the antigen or hapten can be absorbed by a suitable coating on the particles, or it may be chemically coupled thereto by a bridging agent. In a few instances, an antigen or hapten can be coupled directly to the polymer of the latex particle itself.
  • the method of preparation of the antigen-or hapten-bearing particles is not important, provided that the bound antigen or hapten is accessible to antibody.
  • Protein antigens may suitably be coupled to particles using the process described in our U.K. patent specification No. 2013688A.
  • an antigen When an antigen is to be assayed, it is not necessary for the particles to bear whole antigen: they may instead carry only fragments of the whole antigen which bear the antigenic determinant.
  • the antibody used in the method of the invention may be raised in conventional manner.
  • the agglutinator is preferably rheumatoid factor (RF), but other agglutinators such as a component of complement called C1q, mouse serum and ascitic fluid, can be used. These agglutinators are described in our patent specifications referred to hereinbefore. In the assay of haptens, the agglutinator functions to cause agglutination of those particles on which antibody has become bound to the hapten. The other particles, bearing hapten alone, are not agglutinated. The agglutinator will not react to any significant extent with the free hapten-antibody complexes.
  • RF rheumatoid factor
  • agglutination In the case of antigens, some agglutination would sometimes occur without the use of any agglutinator, but the agglutinator acts as a cross-linker to enhance and increase agglutination. Since, in the assay of antigens, the agglutinator will also react with the free antigen-antibody complexes, sufficient agglutinator should then be used to ensure adequate enhancement of the desired agglutination.
  • interferences can arise particularly (in the case of liquids of biological origin) from non-specific protein-protein interactions.
  • such interactions can be reduced by the use of chaotropic agents, and such agents can be used in the method of the present invention.
  • Another technique for avoiding protein interferences involves treating the liquid sample (prior to assay) with a protease enzyme (e.g. pepsin) to digest the unwanted or interfering proteins. This technique can also be used when assaying protein antigens as is descirbed in our European patent application No. 0051985.
  • the presence and/or amount of the antigen or hapten under assay is determined from the presence and/or extent of agglutination and by reference to standard results.
  • Such selective counting can be effected automatically using for example, a Technicon Autocounter system or similar system. (The words "Technicon” and “Autocounter” are trade marks). Whilst selective counting is preferred, an assessment of the extent of agglutination can be made in other ways, such as by separating the agglutinates from the unagglutinated particles and weighing or otherwise measuring the quantities.
  • the method of the invention can be effected manually or by a continuous automated procedure such as the Technicon PACIA@ technique.
  • Glycine buffered saline GSS
  • 0.1 M NaCI 0.17 mol/I glycine
  • 0.04 g/I Na azide pH adjusted to 9.2 with NaOH.
  • GBS-BSA 10 g/I bovine serum albumin (Biograde from Calbiochem, San Diego, CA) in GBS.
  • HCI-pepsin 0.15 mol/I HCI containing 4 g/I pepsin twice crystallised (from Sigma Chemical Co., St. Louis, Mo.). This solution kept at 0-4°C is stable about one day.
  • IgG anti-digoxin IgG was extracted by Rivanol precipitation from a rabbit antiserum prepared against a digoxin-bovine fibrinogen conjugate. 1 ml of this antiserum was sufficient for 250,000 assays.
  • the anti-digoxin IgG was mixed with RF and GBS-BSA to reach a final titer of 1/50 for RF and 1/4000 of the concentration of IgG in the starting antiserum.
  • the mixture was stored at -20°C in 1 ml aliquots, enough for 60 assays.
  • Latex 0.8 p carboxylated polystyrene particles (10% w/v-Estapor@ K150, lot No. 314).
  • Digoxin-BSA-latex 500 ⁇ l of carboxylated latex were washed in 5 ml of 0.02 mol/I borate buffered saline, pH 8.1 (BBS), centrifuged, resuspended in 1 ml BBS, and activated by 45 min stirred incubation at room temperature with 25 mg of 1-ethyl-3 (3-dimethyl-amino-propyl)-carbodiimide HCI.
  • the activated latex was incubated over-night at 4°C under gentle agitation with 25 11 1 of a 10 g/I solution of BSA-digoxin conjugate in 9 g/I saline. After addition of 250 pl of GBS-BSA the particles were centrifuged, washed three times with GBS-BSA, resuspended in 10 ml GBS-BSA, and sonicated for a few seconds in a Branson Sonifier B12. If to be used within 3 weeks, the digoxin-BSA-latex was stored at 4°C. For longer storage, the reagent was lyophilised without any further treatment. Before use, the latex suspension was diluted 1/4 with GBS containing 5 mM EDTA, 60 g/I polyethylene glycol 6000 (Merck, Darmstadt, West Germany) and NaCI at half saturation.
  • Digoxin was dissolved in ethanol to a concentration of 1 g/I. To prepare the standards, this solution was then diluted with a pool of normal human sera.
  • Chylomicrons usually do not interfere in the latex counting. However, when the serum samples have been frozen, some particles formed by denatured lipoproteins can be counted as if they were latex particles. As most of the present work was done on sera which had been frozen, they were first treated to eliminate this possible source of interference. Samples of about 200 pl were mixed with 100 ul of Freon@ 113 (Serva, Heidelberg, W. Germany) while vortexing and then centrifuged for 5 min at 5000 rev/min.
  • the system comprises essentially four components, a DIAS@ component 1, a pump 2, an autocounter 3 and an electronics module 4.
  • the DIAS component 1 (DIAS@ means diluter-incubator-agitator-sample) comprises a horizontal circular tray 10 mounted for rotation about a vertical axis, and having a series of apertures therein receiving sample tubes 11.
  • the tubes 11 depend below tray 10 into a temperature-controlled chamber.
  • the final reaction mixture is sampled and the sample passes through a manifold in which it may be further diluted and debubbled, for example, and via pump 2 to autocounter 3 for counting unagglutinated particles only.
  • Module 4 prints out, or otherwise indicates, the results.
  • DIAS@ component 1 the sample tubes 11 are agitated to keep the particles in suspension.
  • An unmeasured quantity, approximately 200 ⁇ l, of treated serum was pipetted into a sample tube 11 and placed in the inner row of the sampler tray.
  • a probe from station 12 aspirates 100 ⁇ l of the sample into another sample tube (called a reaction tube), when 15 ⁇ l of the agglutinating mixture and 15 ⁇ l of latex conjugate were sequentially added at stations 13 and 14.
  • the suspension was diluted (station 15) by addition of 0.88 ml of GBS and 88 pl was aspirated (station 16) into a manifold in which the suspension was further diluted 20 times with GBS containing 1 ml/I Tween@ 20. After debubbling, the resulting stream passed through the optical cell counter (3).
  • the concentration of unagglutinated particles was expressed by peak heights on the recorder. Fifty analyses/h were performed on this apparatus.
  • the preliminary pepsin digestion of the samples was done manually.
  • RIA was performed with IMMOPHASE@ kits from Corning Medical, Corning Glass Works, Medfield, Massachusetts, U.S.A.
  • the starting solutions of digoxin were prepared in a pool of normal sera and 1 vol was added to 9 vol of each of the various sera to be tested. The recovery was calculated on 10 different sera for each concentration of digoxin. The first three groups were made with the same 10 sera, whereas the seven other groups were made with different sera.
  • TSH Thyrotropin
  • TSH may be assayed according to the invention.
  • the latex particles are coated with a crude pituitary extract containing TSH, and made up in suspension in PBS-BSA buffer.
  • Antibody to TSH was raised in rabbits, and rheumatoid factor was used as the agglutinator.
  • the standard curve produced is shown in Figure 4 of the drawings. The following protocol was used:

Claims (10)

1. Un procédé d'analyse d'un antigène ou d'un haptène dans un échantillon liquide qui comprend le mélange avec l'échantillon liquide de (i) des particules solides finement divisées portant un réactif, (ii) un agglutinateur et (iii) au moins un autre réactif, et la détermination de la présence et/ou de la quantité dudit antigène ou haptène à partir de l'importance de l'agglutination des particules, caractérisé en ce que:
a) les particules finement divisées portent le même antigène ou un fragment du même antigène portant le déterminant antigénique, ou le même haptène, que dans l'analyse, ou un analogue spécifique de celui-ci;
b) ledit autre réactif est un anticorps contre ledit antigène ou haptène; et où
c) la quantité dudit anticorps ajoutée au mélange est inférieure à la quantité nécessaire pour provoquer une agglutination de 100% des particules.
2. Un procédé selon la revendication 1, caractérisé en ce que: la quantité d'anticorps est telle qu'elle provoque l'agglutination de 40 à 80% des particules.
3. Un procédé selon la revendication 1 ou 2, caractérisé en ce que: l'importance de l'agglutination est mesurée par comptage des particules non agglutinées.
4. Un procédé selon la revendication 1, 2 ou 3, caractérisé en ce que: on analyse un haptène, l'haptène étant un médicament ou un métabolite de médicament, un messager intracellulaire, une hormone, une vitamine ou un haptène d'origine bactérienne ou virale.
5. Un procédé selon l'une quelconque des revendications précédentes, caractérisé en ce que: l'échantillon liquide est un liquide de l'organisme de l'homme ou d'un animal.
6. Un procédé selon l'une quelconque des revendications précédentes, caractérisé en ce que: l'agglutineur est le FR, C1q, du sérum de souris ou du liquide d'ascite de souris.
7. Un procédé selon l'une quelconque des revendications précédentes, caractérisé en ce que: les particules ont une taille d'au plus d'environ 15 pm.
8. Un procédé selon l'une quelconque des revendications précédentes, caractérisé en ce que l'antigène ou l'haptène à analyser est une non-protéine, mais l'échantillon liquide contient une ou plusieurs protéines capables d'interférer dans l'analyse, caractérisé en ce que: on ajoute une enzyme de type protéase à l'échantillon liquide pour digérer le ou lesdites protéines afin d'éliminer leurs effets d'interférence.
9. Un procédé selon l'une quelconque des revendications 1 à 7, dans lequel l'antigène à analyser est une protéine et l'échantillon liquide contient aussi une ou plusieurs autres protéines pouvant interférer dans l'essai, caractérisé en ce que: on ajoute une enzyme de type protéase à l'échantillon liquide pour en digérer les protéines, puis on analyse un fragment caractéristique de l'antigène en utilisant un anticorps réagissant avec ledit fragment caractéristique.
10. Un procédé selon l'une quelconque des revendications 1 à 7, caractérisé en ce que: on ajoute également audit échantillon un ou plusieurs agents chaotropes pour réduire les effets des interactions non spécifiques protéine-protéine interférant dans ladite analyse.
EP82301265A 1981-03-16 1982-03-12 Test d'agglutination de particules Expired EP0061857B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB8108112 1981-03-16
GB8108112 1981-03-16

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EP0061857A1 EP0061857A1 (fr) 1982-10-06
EP0061857B1 true EP0061857B1 (fr) 1985-11-06

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US (1) US4427781A (fr)
EP (1) EP0061857B1 (fr)
JP (1) JPS57206859A (fr)
AU (1) AU548003B2 (fr)
CA (1) CA1174596A (fr)
DE (1) DE3267228D1 (fr)

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EP1950565B1 (fr) 2005-11-18 2011-03-23 Nitto Boseki Co., Ltd. Procédé de dosage d'un antigène
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Publication number Publication date
DE3267228D1 (en) 1985-12-12
JPH05665B2 (fr) 1993-01-06
CA1174596A (fr) 1984-09-18
JPS57206859A (en) 1982-12-18
AU8124782A (en) 1982-09-23
EP0061857A1 (fr) 1982-10-06
US4427781A (en) 1984-01-24
AU548003B2 (en) 1985-11-14

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